Water dynamics around T0 vs R4 of hemoglobin from local hydrophobicity analysis.

The local hydration around tetrameric hemoglobin (Hb) in its T0 and R4 conformational substates is analyzed based on molecular dynamics simulations. Analysis of the local hydrophobicity (LH) for all residues at the α1ß2 and α2ß1 interfaces, responsible for the quaternary T → R transition, which is encoded in the Monod-Wyman-Changeux model, as well as comparison with earlier computations of the solvent accessible surface area, makes clear that the two quantities measure different aspects of hydration. Local hydrophobicity quantifies the presence and structure of water molecules at the interface, whereas "buried surface" reports on the available space for solvent. For simulations with Hb frozen in its T0 and R4 states, the correlation coefficient between LH and buried surface is 0.36 and 0.44, respectively, but it increases considerably if the 95% confidence interval is used. The LH with Hb frozen and flexible changes little for most residues at the interfaces but is significantly altered for a few select ones: Thr41α, Tyr42α, Tyr140α, Trp37ß, Glu101ß (for T0) and Thr38α, Tyr42α, Tyr140α (for R4). The number of water molecules at the interface is found to increase by ∼25% for T0 → R4, which is consistent with earlier measurements. Since hydration is found to be essential to protein function, it is clear that hydration also plays an essential role in allostery.

Hydration dynamics and IR spectroscopy of 4-fluorophenol.

Halogenated groups are relevant in pharmaceutical applications and potentially useful spectroscopic probes for infrared spectroscopy. In this work, the structural dynamics and infrared spectroscopy of para-fluorophenol (F-PhOH) and phenol (PhOH) is investigated in the gas phase and in water using a combination of experiment and molecular dynamics (MD) simulations. The gas phase and solvent dynamics around F-PhOH and PhOH is characterized from atomistic simulations using empirical energy functions with point charges or multipoles for the electrostatics, Machine Learning (ML) based parametrizations and with full ab initio (QM) and mixed Quantum Mechanical/Molecular Mechanics (QM/MM) simulations with a particular focus on the CF- and OH-stretch region. The CF-stretch band is heavily mixed with other modes whereas the OH-stretch in solution displays a characteristic high-frequency peak around 3600 cm-1 most likely associated with the -OH group of PhOH and F-PhOH together with a characteristic progression below 3000 cm-1 due to coupling with water modes which is also reproduced by several of the simulations. Solvent and radial distribution functions indicate that the CF-site is largely hydrophobic except for simulations using point charges which renders them unsuited for correctly describing hydration and dynamics around fluorinated sites. The hydrophobic character of the CF-group is particularly relevant for applications in pharmaceutical chemistry with a focus on local hydration and interaction with the surrounding protein.

Structure, Organization, and Heterogeneity of Water-Containing Deep Eutectic Solvents.

The spectroscopy and structural dynamics of a deep eutectic mixture (KSCN/acetamide) with varying water content is investigated from 2D IR (with the C-N stretch vibration of the SCN- anions as the reporter) and THz spectroscopy. Molecular dynamics simulations correctly describe the nontrivial dependence of both spectroscopic signatures depending on water content. For the 2D IR spectra, the MD simulations relate the steep increase in the cross-relaxation rate at high water content to the parallel alignment of packed SCN- anions. Conversely, the nonlinear increase of the THz absorption with increasing water content is mainly attributed to the formation of larger water clusters. The results demonstrate that a combination of structure-sensitive spectroscopies and molecular dynamics simulations provides molecular-level insights into the emergence of heterogeneity of such mixtures by modulating their composition.

Site-selective dynamics of ligand-free and ligand-bound azidolysozyme.

Azido-modified alanine residues (AlaN3) are environment-sensitive, minimally invasive infrared probes for the site-specific investigation of protein structure and dynamics. Here, the capability of the label is investigated to query whether or not a ligand is bound to the active site of lysozyme and how the spectroscopy and dynamics change upon ligand binding. The results demonstrate specific differences for center frequencies of the asymmetric azide stretch vibration, the longtime decay, and the static offset of the frequency fluctuation correlation function (FFCF)-all of which are experimental observables-between the ligand-free and the ligand-bound N3-labeled protein. The center-frequency shifts range from 1 to 8 cm-1, which is detectable from state-of-the art experiments. Similarly, the nonvanishing static component Δ0 of the FFCF between ligand-free and ligand-bound protein can differ by up to a factor of 2.5. This makes the azide label a versatile and structurally sensitive probe to report on the dynamics of proteins in a variety of environments and for a range of different applications. Ligand-induced differences in the dynamics are also mapped onto changes in the local and through-space coupling between residues by virtue of dynamical cross correlation maps. This demonstrates that the position where the label is placed also influences the local and global protein motions.

Cross-Correlated Motions in Azidolysozyme.

The changes in the local and global dynamics of azide-labelled lysozyme compared with that of the wild type protein are quantitatively assessed for all alanine residues along the polypeptide chain. Although attaching -N3 to alanine residues has been considered to be a minimally invasive change in the protein it is found that depending on the location of the alanine residue, the local and global changes in the dynamics differ. For Ala92, the change in the cross-correlated motions are minimal, whereas attaching -N3 to Ala90 leads to pronounced differences in the local and global correlations as quantified by the cross-correlation coefficients of the Cα atoms. We also demonstrate that the spectral region of the asymmetric azide stretch distinguishes between alanine attachment sites, whereas changes in the low frequency, far-infrared region are less characteristic.

Site-selective dynamics of azidolysozyme.

The spectroscopic response of and structural dynamics around all azido-modified alanine residues (AlaN3) in lysozyme are characterized. It is found that AlaN3 is a positionally sensitive probe for the local dynamics, covering a frequency range of ∼15 cm-1 for the center frequency of the line shape. This is consistent with findings from selective replacements of amino acids in PDZ2, which reported a frequency span of ∼10 cm-1 for replacements of Val, Ala, or Glu by azidohomoalanine. For the frequency fluctuation correlation functions, the long-time decay constants τ2 range from ∼1 to ∼10 ps, which compares with experimentally measured correlation times of 3 ps. Attaching azide to alanine residues can yield dynamics that decays to zero on the few ps time scale (i.e., static component Δ0 ∼ 0 ps-1) or to a remaining, static contribution of ∼0.5 ps-1 (corresponding to 2.5 cm-1), depending on the local environment on the 10 ps time scale. The magnitude of the static component correlates qualitatively with the degree of hydration of the spectroscopic probe. Although attaching azide to alanine residues is found to be structurally minimally invasive with respect to the overall protein structure, analysis of the local hydrophobicity indicates that the hydration around the modification site differs for modified and unmodified alanine residues, respectively.

Dynamics and Infrared Spectrocopy of Monomeric and Dimeric Wild Type and Mutant Insulin.

The infrared spectroscopy and dynamics of -CO labels in wild type and mutant insulin monomer and dimer are characterized from molecular dynamics simulations using validated force fields. It is found that the spectroscopy of monomeric and dimeric forms in the region of the amide-I vibration differs for residues B24-B26 and D24-D26, which are involved in dimerization of the hormone. Also, the spectroscopic signatures change for mutations at position B24 from phenylalanine, which is conserved in many organisms and is known to play a central role in insulin aggregation, to alanine or glycine. Using three different methods to determine the frequency trajectories (solving the nuclear Schrödinger equation on an effective 1-dimensional potential energy curve, using instantaneous normal modes, and using parametrized frequency maps) leads to the same overall conclusions. The spectroscopic response of monomeric WT and mutant insulin differs from that of their respective dimers, and the spectroscopy of the two monomers in the dimer is also not identical. For the WT and F24A and F24G monomers, spectroscopic shifts are found to be ∼20 cm-1 for residues (B24-B26) located at the dimerization interface. Although the crystal structure of the dimer is that of a symmetric homodimer, dynamically the two monomers are not equivalent on the nanosecond time scale. Together with earlier work on the thermodynamic stability of the WT and the same mutants, it is concluded that combining computational and experimental infrared spectroscopy provides a potentially powerful way to characterize the aggregation state and dimerization energy of modified insulins.

Non-conventional force fields for applications in spectroscopy and chemical reaction dynamics.

Extensions and improvements of empirical force fields are discussed in view of applications to computational vibrational spectroscopy and reactive molecular dynamics simulations. Particular focus is on quantitative studies, which make contact with experiments and provide complementary information for a molecular-level understanding of processes in the gas phase and in solution. Methods range from including multipolar charge distributions to reproducing kernel Hilbert space approaches and machine learned energy functions based on neural networks.

Vibrational Spectroscopy of N3- in the Gas and Condensed Phase.

Azido-derivatized amino acids are potentially useful, positionally resolved spectroscopic probes for studying the structural dynamics of proteins and macromolecules in solution. To this end, a computational model for the vibrational modes of N3- based on accurate electronic structure calculations and a reproducing kernel Hilbert space representation of the potential energy surface for the internal degrees of freedom is developed. Fully dimensional quantum bound state calculations yield the antisymmetric stretch vibration at 1974 cm-1 compared with 1986 cm-1 from experiment. This mode shifts by 64 cm-1 (from the frequency distribution) and 74 cm-1 (from the IR line shape) to the blue, respectively, compared with 61 cm-1 from experiment for N3- in water. The decay time of the frequency fluctuation correlation function is 1.1 ps, which is in good agreement with experiment (1.2-1.3 ps) and the full width at half maximum of the asymmetric stretch in solution is 18.5 cm-1 compared with 25.2 cm-1 from experiment. A computationally more efficient analysis based on instantaneous normal modes is shown to provide comparable, albeit somewhat less quantitative results compared to solving the three-dimensional Schrödinger equation for the fundamental vibrations.